Arijit Roy

Mice lacking in gp91 phox subunit of NAD(P)H oxidase showed glomus cell [Ca2+]i and respiratory responses to hypoxia

The hypothesis that NAD(P)H oxidase may serve as an oxygen sensor was tested using the mice deficient (knock-out) in gp91phox subunit of NAD(P)H oxidase enzyme complex and compared with wild-type (C57BL/6J) strain measuring the ventilatory and glomus cell intracellular calcium ([Ca(2+)](i)) responses of carotid body to hypoxia. The hypoxic ventilatory responses as well as the [Ca(2+)](i) were preserved in the NAD(P)H oxidase knock-out mice. NAD(P)H oxidase, though a major source of oxygen radical production, is not the oxygen sensor in mice carotid body.

Regulation of oxygen sensing in peripheral arterial chemoreceptors

The carotid bodies are a small pair of highly vascularized and well perfused organs located at each carotid artery bifurcation, strategically situated to sense oxygen in arterial blood as it leaves the heart. Carotid body glomus cells are identified as the primary oxygen sensors, which respond to changes in blood P(O(2)) within milliseconds. Acute hypoxia causes a rapid increase in carotid sinus nerve (CSN) activity, providing afferent signals to the respiratory center in the brainstem. Glomus cells secrete numerous neurotransmitters that modulate CSN firing rates. This review will discuss major hypotheses that have emerged regarding acute oxygen sensing by glomus cells. In contrast, chronic responses to hypoxia are much slower, involving cytosolic reactions that take place over several minutes and nuclear reactions which occur over several hours. Converging concepts from different areas of research in oxygen sensing cells and tissues (including the carotid body) have been combined to describe molecular and biochemical changes that take place in the carotid body with chronic hypoxia. These include oxygen dependent proteolytic processes in the cytosol and gene transcription in the nucleus. In addition, cellular and nuclear responses to chronic hypoxia will be discussed.

K+-current modulated by PO2 in type I cells in rat carotid body is not a chemosensor

According to the membrane channel hypothesis of carotid body O2 chemoreception, hypoxia suppresses K+ currents leading to cell depolarization, [Ca2+]i rise, neurosecretion, increased neural discharge from the carotid body. We show here that tetraethylammonium (TEA) plus 4-aminopyridine (4-AP) which suppressed the Ca2+ sensitive and other K+ currents in rat carotid body type I cells, with and without low [Ca2+]o plus high [Mg2+]o, did not essentially influence low PO2 effects on [Ca2+]i and chemosensory discharge. Thus, hypoxia may suppress the K+ currents in glomus cells but K+ current suppression of itself does not lead to chemosensory excitation. Therefore, the hypothesis that K+-O2 current is linked to events in chemoreception is not substantiated. K+-O2 current is an epiphemenon which is not directly linked with O2 chemoreception.

Lessons from chronic intermittent and sustained hypoxia at high altitudes.

Recurrent sleep apnea (RSA), mimicking chronic intermittent hypoxia (CIH), may trigger unique adaptations in oxygen sensing in the carotid body, and consequent cellular functions unlike the effects of sustained hypoxia (SH). As a mechanism, an augmented generation of reactive oxygen species (ROS) in CIH has been invoked at the exclusion of SH effects. The ROS might act at hypoxia inducible factors (HIF-1s), giving rise to various genes whose function is to restore the tissue P(O(2)) close to the original. In a spate, review articles on the CIH effects at sea level have appeared but little on high altitude (HA). Their views have been reexamined with the primary focus on the peripheral chemoreception. At HA, RSA is more common in the lowlanders because of a high ventilatory sensitivity to hypoxia (with the consequent effects) unlike the high altitude natives (HAN). Undoubtedly, the HIF-1s play a central role at HA, the mechanisms of which are unknown and explorable.

PO2-PCO2 stimulus interaction in [Ca2+]i and CSN activity in the adult rat carotid body

Since glomus cell intracellular calcium ([Ca(2+)](i)) plays a key role in generating carotid sinus nerve (CSN) discharge, we hypothesized that glomus cell [Ca(2+)](i) would correspond to CSN discharge rates during P(O(2))-P(CO(2)) stimulus interaction in adult rat carotid body (CB). Accordingly, we measured steady state P(O(2))-P(CO(2)) interaction in CSN discharge rates during hypocapnia (P(CO(2))=8-10 Torr), normocapnia (P(CO(2))=33-35 Torr) and hypercapnia (P(CO(2))=68-70 Torr) in normoxia (P(O(2)) approximately 130 Torr) and hypoxia (P(O(2)) approximately 36 Torr). The results showed P(O(2))-P(CO(2)) stimulus interaction in CSN responses. [Ca(2+)](i) levels were measured in isolated type I cells (2-3 cells/field), using Ca(2+) sensitive fluoroprobe indo-1AM. The [Ca(2+)](i) responses increased with increasing P(CO(2)) in normoxia. In hypoxia, [Ca(2+)](i) did not increase during hypocapnia but increased during normocapnia, showing P(O(2))-P(CO(2)) interaction. However, CSN response during hypoxia was far greater than that for [Ca(2+)](i) response, particularly during hypocapnic hypoxia. Thus, the [Ca(2+)](i) interaction cannot account for the whole CSN interaction. The origin of this CSN P(O(2)-)P(CO(2)) interaction must have occurred in part beyond cellular [Ca(2+)](i) interaction. Interactions at both sites (glomus cell membrane and sinus nerve endings) are reminiscent of reversible O(2)-heme protein reaction with a Bohr effect.

Indirect 17O-magnetic resonance imaging of cerebral blood flow in the rat

Proton T1ρ‐dispersion MRI is demonstrated for indirect, in vivo detection of 17O in the brain. This technique, which may be readily implemented on any clinical MRI scanner, is applied towards high‐resolution, quantitative mapping of cerebral blood flow (CBF) in the rat by monitoring the clearance of 17O‐enriched water. Strategies are derived and employed for 1) quantitation of absolute H217O tracer concentration from a ratio of high‐ and low‐frequency spin‐locked T1ρ images, and 2) mapping CBF without having to transform the T1ρ signal to H217O tracer concentration. Absolute regional blood flow was mapped in a single 3‐mm brain slice at an in‐plane resolution of 0.4 × 0.8 mm within a 5‐min tracer washout time; these data are consistent with the less localized CBF measurements reported in the literature. T1ρ‐weighted imaging yields excellent signal‐to‐noise ratios, spatiotemporal resolution, and anatomical contrast for mapping CBF. Magn Reson Med 49:479–487, 2003.

Purines, the carotid body and respiration

The carotid body is essential to detecting levels of oxygen in the blood and initiating the compensatory response. Increasing evidence suggests that the purines ATP and adenosine make a key contribution to this signaling by the carotid body. The glomus cells release ATP in response to hypoxia. This released ATP can stimulate P2X receptors on the carotid body to elevate intracellular Ca(2+) and to produce an excitatory response. This released ATP can be dephosphorylated to adenosine by a series of extracellular enzymes, which in turn can stimulate A(1), A(2A) and A(2B) adenosine receptors. Levels of extracellular adenosine can also be altered by membrane transporters. Endogenous adenosine stimulates these receptors to increase the ventilation rate and may modulate the catecholamine release from the carotid sinus nerve. Prolonged hypoxic challenge can alter the expression of purinergic receptors, suggesting a role in the adaptation. This review discusses evidence for a key role of ATP and adenosine in the hypoxic response of the carotid body, and emphasizes areas of new contributions likely to be important in the future.

High PCO does not alter pHi, but raises [Ca2+]i in cultured rat carotid body glomus cells in the absence and presence of CdCl2

We measured the effect of high PCO (500-550 Torr) on the pHi and [Ca2+]i in cultured glomus cells of adult rat carotid body (CB) as a test of the two models currently proposed for the mechanism of CB chemoreception. The metabolic model postulates that the rise in glomus cell [Ca2+]i, the initiating reaction in the signalling pathway leading to chemosensory neural discharge, is due to [Ca2+] release from intracellular Ca2+ stores. The membrane potential model postulates that the rise in [Ca2+]i comes from influx of extracellular Ca2+ through voltage-dependent Ca2+ channels (VDCC) of the L-type. High PCO did not change pHi at PO2 of 120-135 Torr, showing that CO-induced changes in [Ca2+]i are not due to changes in pHi. High PCO caused a highly significant rise in [Ca2+]i from 90+/-12 nM to 675+/-65 nM, both in the absence and in the presence of 200 microM CdCl2, a potent blocker of L-type VDCCs. This result is fully consistent with release of Ca2+ from glomus cell intracellular stores according to metabolic model, but inconsistent with influx of extracellular Ca2+ through VDCCs according to the membrane potential model.

Activation of HIF-1α mRNA by hypoxia and iron chelator in isolated rat carotid body

Abstract The hypoxia inducible factor-1α (HIF-1α) protein level is increased by hypoxia and iron chelator (ciclopirox olamine) in isolated rat carotid body (CB) and glomus cells. Reverse transcription and polymerase chain reaction (RT-PCR) are performed to test whether this increase is caused, at least in part, by increased HIF-1α gene transcription. HIF-1α mRNA levels dose-dependently increased and decreased in the rat CBs incubated for 1 h in a medium saturated with O 2 levels that were varied around nominally normoxic level of 21% in the 0–95% range. The iron chelator, ciclopirox olamine (5 μM), stimulated HIF-1α mRNA production under normoxic condition. Thus, in the CB, the main systemic O 2 -sensing organ, HIF-1α transcription is regulated by O 2 supply around the normoxic level; this may contribute to cellular and organismal adaptations to chronic changes in ambient O 2 .

Chemosensory response to high pCO is blocked by cadmium, a voltage-sensitive calcium channel blocker

In the dark, during normocapnic (pCO2=35 Torr, pHo=7.4) normoxia (pO2=100 Torr), high pCO (>300 Torr) causes Ca2+-dependent photolabile excitation of chemosensors in the carotid body (CB). We previously proposed that the source of this Ca2+ was the [Ca2+]i stores because CO would react only intracellularly. However, influx of extracellular Ca2+ was not excluded. Now, using perfused rat CB (n=6) in the presence of normal extracellular [Ca2+] we show that chemosensory response to CO (pCO approximately 550 Torr) in normoxic (pO2 approximately 100 Torr) normocapnia (pCO2 approximately 30 Torr, pH approximately 7.4) is completely but reversibly inhibited by Cd2+ (200 microM), a voltage-gated Ca2+ channel blocker. Thus, extracellular Ca2+ is necessary for excitatory chemosensory response to high pCO. Cd2+ block occurs in spite of an enhanced [Ca2+]i rise. This shows that Ca2+ rise alone is unable to release neurotransmitter and to elicit a chemosensory response. Therefore, as a corollary, we conclude that Cd2+ blocks the Ca2+ flux that is needed for vesicle-membrane fusion for neurotransmitter release and neural discharge.